1. Field of the Invention
The present invention relates generally to the fields of organic synthesis of bioactive compounds. More particularly, it concerns the chemical method of synthesis for N-homocysteine thiolactonyl retinamide, a compound that has anticancer and antiatherogenic properties.
2. Description of Related Art
Chemotherapeutic agents such as cyclophosphamide which are commonly used as antineoplastic agents have the disadvantage of cumulative toxicity after prolonged administration in cancer chemotherapy. The subject compound of this invention, N-homocysteine thiolactonyl retinamide, overcomes the disadvantage of toxicity of chemotherapeutic compounds and retinoids, because it is composed of retinoic acid and homocysteine thiolactone bound together in a non-toxic form. Because large doses of N-homocysteine thiolactonyl retinamide can be given without toxicity, the compound is useful for chemoprevention and chemotherapy of malignant neoplasms in animals and for use as an anti-atherogenic agent.
Homocysteine thiolactone is produced naturally in the liver as a metabolite of the essential amino acid, methionine (Spindel and McCully, 1974). Anticancer properties have been demonstrated for several homocysteine thiolactone derivatives (U.S. Pat. Nos. 4,255,443; 4,383,994; 4,618,685; McCully and Vezeridis 1985; McCully and Vezeridis 1987a,b; McCully and Clopath, 1977; McCully 1976; McCully, 1994b).
Retinoids are another group of chemicals which have been demonstrated to have chemopreventive activity against a variety of carcinogens (Moon & Itri, 1984). They are also needed in the reaction of homocysteine with oxygen (Sundaresen, 1966). These observations supported the rational of conjugating homocysteine thiolactone with retinoic acid in an N-substituted fashion to examine its influence on cancerous cells. The synthesis of N-homocysteine thiolactonyl retinamide (also called thioretinamide) from homocysteine thiolactone free base and retinoic acid was attempted by McCully in U.S. Pat. No. 4,618,685.
In general, the procedure described in U.S. Pat. No. 4,618,685 by McCully in 1986, incorporated herein by reference, involves the conjugation of homocysteine thiolactone to retinoic acid using dicyclohexylcarbodiimide as a coupling agent. As specifically described in that patent, the process starts with the preparation of the free base of homocysteine thiolactone and involves the dissolution of 1.01 g of sodium hydroxide in 25 ml water, followed by the addition of 100 ml of methylene chloride, accompanied with rapid mixing, and the further addition of 3.84 g of homocysteine thiolactone hydrochloride slowly to the mixture. After 15 minutes of mixing, the methylene chloride layer is separated, dried over anhydrous sodium sulfate and the solvent is evaporated under reduced pressure at 37.degree. C. 1.17 g (10 mmoles) of the resulting clear liquid (homocysteine thiolactone free base) is immediately added to 50 ml of tetrahydrofuran (or any other non-polar solvent) containing 3.00 g (10 mmoles) of all-trans-retinoic acid. Then, 2.06 g (10 mmoles) of dicyclohexylcarbodiimide is added, and the reaction mixture is stirred 16 hours at 20.degree. C., protected from light. The tetrahydrofuran is removed at 37.degree. C. under reduced pressure, and the yellow-white residue is added to 500 ml of water and 500 ml of ethyl acetate. The mixture is stirred vigorously for one hour, and the ethyl acetate layer is separated and dried over anhydrous sodium sulfate. The ethyl acetate is concentrated to about 20 ml at 50.degree. C. under reduced pressure and cooled. 2.75 g of yellow powder (N-homocysteine thiolactonyl retinamide) represents 69% of theoretical yield. m.p. 172.degree. C. Analysis: C, calculated, 72.2; found, 71.99, corresponds to C.sub.24 H.sub.33 NSO.sub.2. NMR 60 MHz multiplets 1.1-2.2, triplet 6.3, singlet 7.2 ppm.
Unfortunately, it has recently been shown that the method used to produce homocysteine thiolactonly retinamide in U.S. Pat. No. 4,618,685 does not produce the desired compound in pure form. The inventors of the present invention attempted the method of U.S. Pat. No. 4,618,685, and McCully and Vezeridis, 1987a, and were unsuccessful in obtaining sufficient, pure thioretinamide as confirmed by subsequent analysis by 300 MHz N.M.R. Proton N.M.R. analysis of the compounds produced by McCully's method clearly showed production of dicyclohexylurea (DCU), an unwanted byproduct of the reaction and hence the purity of thioretinamide produced was reduced. In sum, the procedure described in the above-listed references did not produce thioretinamide in the expected quantity or purity.
Despite the difficulties in synthesizing thioretinamide following the procedures described in the McCully patent, the work of McCully showed that there was promise for thioretinamide as a therapeutic. The "thioretinamide" synthesized as described was able to counteract the carcinogenecity of ethyl carbamate in pulmonary neoplasms of strain A female mice. Thioretinamide decreased the number of tumors formed to 80% at doses of 50 mg/week, and 60% at doses of 200 mg/week. Unfortunately, the 200 mg/week mice showed significant weight loss (McCully and Vezeridis, 1987a). Additionally, in U.S. Pat. No. 4,925,931, incorporated herein by reference, thioretinamide has been shown to react with cobalamin to form N-homocysteine thiolactonyl retinamido cobalamin, also known as thioretinaco. Both thioretinamide and thioretinaco have anticarcinogenic and antineoplastic activities, as reported in McCully and Vezeridis, 1987a and McCully and Vezeridis, 1989. In experiments with cultured malignant and normal cells, thioretinaco was found to have antiproliferative activity, and thioco, the complex of homocysteine thiolactone and cobalamin, was found to increase growth of both malignant and normal cells, as reported in McCully et al., 1992. Intra-tumoral administration of thioretinaco decreased the growth of human pancreatic adenocarcinomas in athymic mice, as reported in McCully et al., 1989.
The above-described studies and patents point to great promise for thioretinamide as a chemotherapeutic agent. However, in order to fully realize the potential of this agent, a method of producing it in sufficient quantities and purity is needed. Thus, there is need for the development of methods that produces high yields of pure N-homocysteine thiolactonyl retinamide.